FMRFamide (Phe-Met-Arg-Phe amide) and related peptides comprise a family of neuropeptides that are abundant in many invertebrates, including Caenorhabditis elegans (Nelson, L. S., Kim, K., Memmott, J. E., and Li, C. (1998). FMRFamide-related gene family in the nematode, Caenorhabditis elegans. Mol Brain Res 58, 103-111), Aplysia californica (Greenberg, M. J., and Price, D. A. (1992). Relationships among the FMRFamide-like peptides. Prog Brain Res. 92, 25-37), and Drosophila melanogaster (Schneider, L. E., and Taghert, P. H. (1988). Isolation and characterization of a Drosophila gene that encodes multiple neuropeptides related to Phe-Met-Arg-Phe-NH2 (FMRF amide). Proc Natl Acad Sci USA 85, 1993-1997). In these organisms, FMRFamide-like neuropeptides act as neurotransmitters and neuromodulators. At least one gene encoding FMRFamide-related peptides is present in mammals; it produces neuropeptide FF and neuropeptide AF (A18Famide) (Perry, S. J., Huang, E. Y. K., Cronk, D., Bagust, J., Sharma, R., Walker, R. J., Wilson, S., and Burke, J. F. (1997). A human gene encoding morphine modulation peptides related to NPFF and FMRF amide, FEBS Lett 409, 426-430; Vilim, F. S., Aarnisalo, A. A., Nieminen, M. L., Lintunen, M., Karlstedt, K., Kontinen, V. K., Kalso, E., States, B., Panula, P., and Ziff, E. (1999). Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli. Mol Pharmacol 55, 804-811). Although FMRFamide itself has not been discovered in mammals (Yang, H. Y. T., Fratta, W., Majane, E. A., and Costa, E. (1985). Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 82, 7757-7761), administration of FMRFamide induces a variety of physiologic effects, including alterations in blood pressure, respiratory rate, glucose-stimulated insulin release, and behavior (Kavaliers, G. M., and Hirst, M. (1985). FMRFamide, a putative endogenous opiate antagonist: evidence from suppression of defeat-induced analgesia and feeding in mice. Neuropeptides 6, 485-494; Kavaliers, M. (1987). Calcium channel blockers inhibit the antagonistic effects of Phe-Met-Arg-Phe-amide (FMRFamide) on morphine- and stress-induced analgesia in mice. Brain Res 415, 380-384; Mues, G., Fuchs, I., Wei, E. T., Weber, E., Evans, C. J., Barchas, J. D., and Chang, J.-K. (1982). Blood pressure elevation in rats by peripheral administration of Tyr-Gly-Gly-Phe-Met-Arg-Phe and the invertebrate neuropeptide, Phe-Met-Arg-Phe-NH2. Life Sciences 31, 2555-2561; Muthal, A. V., Mandhane, S. N., and Chopde, C. T. (1997). Central administration of FMRFamide produces antipsychotic-like effects in rodents. Neuropeptides 31, 319-322; Nishimura, M., Ohtsuka, K., Takahashi, H., and Yoshimura, M. (2000). Role of FMRFamide-Activated Brain Sodium Channel in Salt-Sensitive Hypertension. Hypertension 35, 443-450; Raffa, R. B., Heyman, J., and Porreca, F. (1986) Intrathecal FMRFamide (Phe-Met-Arg-Phe-NH2) induces excessive grooming behavior in mice. Neuroscience Lett 65, 94-98; Sorenson, R. L., Sasek, C. A., and Elde, R. P. (1984). Phe-Met-Arg-Phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet. Peptides 5, 777-782; Tekegdy, G., and Bollxc3x3k, I. (1987). Amnesic action of FMRFamide in rats. Neuropeptides 10, 157-163; Thiemermann, C., Al-Damluji, S., Hecker, M., and Vane, J. R. (1991). FMRF-amide and L-Arg-1-Phe increase blood pressure and heart rate in the anaesthetized rate by central stimulation of the sympathetic nervous system. Biochem Biophys Res Comm 175, 318-324). In mammals, FMRFamide and neuropeptide FF also modify the response to painful stimuli and are induced by inflammation (Kontinen, V. K., Aarnisalo, A. A., Idanpaan-Heikkila, J. J., Panula, P., and Kalso, E. (1997). Neuropeptide FF in the rat spinal cord during carrageenan inflammation. Peptides 18, 287-292; Raffa, R. B., and Connelly, C. D. (1992). Supraspinal antinociception produced by [D-Met2]-FMRFamide in mice. Neuropeptides 22, 195-203; Tang, J., Yang, H. Y. T., and Costa, E. (1984). Inhibition of spontaneous and opiate-modified nociception by an endogenous neuropeptide with Phe-Met-Arg-Phe-NH2-like immunoreactivity. Proc Natl Acad Sci USA 81, 5002-5005; Vilim, F. S., Aarnisalo, A. A., Nieminen, M. L., Lintunen, M., Karlstedt, K., Kontinen, V. K., Kalso, E., States, B., Panula, P., and Ziff, E. (1999). Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli. Mol Pharmacol 55, 804-811; Yang, et al. (1985)). When FMRFamide and related peptides are injected intracerebroventricularly, they elicit hyperalgesia and a reduction in morphine-induced analgesia (Brussard, A. B., Kits, K. S., Ter Maat, A., Mulder, A. H., and Schoffelmeer, A. N. M. (1989). Peripheral injection of DNA-RFa, a FMRFa agonist, suppresses morphine-induced analgesia in rats. Peptides 10, 735-739; Kavaliers (1987); Raffa, R. B. (1988). The action of FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides on mammals. Peptides 9, 915-922; Roumy, M., and Zajac, J. M. (1998). Neuropeptide FF, pain and analgesia. Euro J. Pharm 345, 1-11; Tang et al. (1984); Yang, et al. (1985)). In addition, FMRFamide immunoreactive material is released in mammals following chronic morphine administration, and anti-FMRFamide antibodies can enhance morphine""s effects (Devillers, J. P., Boisserie, F., Laulin, J. P., Larcher, A., and Simonnet, G. (1995). Simultaneous activation of spinal antiopioid system (neuropeptide FF) and pain facilitatory circuitry by stimulation of opioid receptors in rats. Brain Research 700, 173-181; Tang, et al. (1984)).
Some effects of FMRFamide and neuropeptide FF appear to be mediated through opioid receptors; these effects are blocked by the opioid antagonist naloxone (Gouardxc3xa9res, C., Sutak, M., Zajak, J. M. and Jhamandas, K. (1993). Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat. Eur J Pharm 237, 73-81; Kavaliers and Hirst (1985); Kavaliers (1987); Raffa (1988); Roumy and Zajac (1998)). Yet other effects of FMRFamide and FMRFamide-related peptides are independent of opioid receptors and are insensitive to naloxone (Allard, M., Geoffre, S., Legendre, P., Vincent, J. D., and Simonnet, G. (1989). Characterization of rat spinal cord receptors to FLFQPQRFamide, a mammalian morphine modulating peptide: a binding study. Brain Research 500, 169-176; Gayton, R. J. (1982). Mammalian neuronal actions of FMRFamide and the structurally related opioid Met-enkephalin-Arg6-Phe7. Nature 298, 275-176; Kavaliers (1987); Raffa (1988); Raffa, et al. (1986); Roumy and Zajac (1998)). In mammals, the non-opioid receptor(s) for FMRFamide and related peptides have not been identified, and it is not known how these peptides modulate pain sensation. However, the discovery of a FMRFamide-activated Na+ channel (FaNaCh) in the mollusc Heix aspersa (Lingueglia, E., Champigny, G., Lazdunski, M., and Barbry, P. (1995). Cloning of the amiloride-sensitive FMRFamide peptide-gated sodium channel. Nature 378, 730-733) provided a clue that similar receptors might exist in mammals.
Unlike many neuropeptide receptors, FaNaCh is an ion channel gated directly by its peptide ligand, FMRFamide (Lingueglia, et al. (1995)). The neuropeptide receptor, FaNaCh, is a member of the DEG/ENaC family of channels. DEG/ENaC channels are homo- or hetero-multimers composed of multiple subunits (Bassilana, F., Champigny, G., Waldmann, R., de Weille, J. R., Heurteaux, C., and Lazdunski, M. (1997). The acid-sensitive ionic channel subunit ASIC and the mammalian degenerin MDEG form a heteromultimeric H+-gated Na+ channel with novel properties. J. Biol Chem 272, 28819-28822; Coscoy, S., Lingueglia, E., Lazdunski, M., and Barbry, P. (1998). The Phe-Met-Arg-Phe-amide-activated sodium channel is a tetrameter. J Biol Chem 273, 8317-8322; Lingueglia, E., de Weille, J. R., Bassilana, F., Heurteaux, C., Sakai, H., Waldmann, R., and Lazdunski, M. (1997). A modulatory subunit of acid sensing ion channels in brain and dorsal root ganglion cells. J Biol Chem 272, 29778-29783; Waldmann, R., and Lazdunski, M. (1998). H+-gated cation channels: neuronal acid sensors in the NaC/DEG family of ion channels. Curr Opin Neurobiol 8, 418-424). Each subunit contains two transmembrane domains separated by a large extracellular cysteine-rich domain, and cytosolic N- and C-termini (Waldmann and Lazdunski (1998)). DEG/ENaC channels are not voltage-gated and are cation-selective (usually Na+ greater than K+). FaNaCh is the only known DEG/ENaC channel which acts as a neuropeptide receptor. Other members of this family are involved in mechanosensation, salt taste, and epithelial Na30  absorption (Lindemann, B. (1996). Taste reception. Physiol Rev 76, 718-766; Mano, I., and Driscoll, M. (1999). DEG/ENaC channels: a touchy superfamily that watches its salt. Bioessays 21, 568-578; Schild, L., Canessa, C. M., Shimkets, R. A., Gautschi, I., Lifton, R. P., and Rossier, B. C. (1995). A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci USA 92, 5699-5703; Snyder, P. M., Price, M. P., McDonald, F. J., Adams, C. M., Volk, K. A., Zeiher, B. G., Stokes, J. B., and Welsh, M. J. (1995). Mechanism by which Liddle""s syndrome mutations increase activity of a human epithelial Na+ channel. Cell 83, 969-978). Although a mammalian FaNaCh has not yet been isolated, mammals do possess multiple DEG/ENaC family members. Interestingly, one subset of this channel family, the acid-sensing ion channels, has been postulated to play a role in sensory perception and may, like FMRFamide, play a role in pain perception (Waldmann and Lazdunski (1998)). The acid-sensing DEG/ENaC channels respond to protons and generate a voltage-insensitive cation current when the extracellular solution is acidified.
The tissue acidosis associated with inflammation, infection, and ischemia causes pain (Reeh, P. W., and Steen, K. H. (1996). Tissue acidosis in nociception and pain. Prog Brain Res 113, 143-151). Acidosis also generates proton-dependent transient and sustained Na30  currents in cultured sensory neurons (Bevan, S., and Yeats, J. (1991). Protons activate a cation conductance in a sub-population of rat dorsal root ganglion neurones. J Physiol (Lond) 433, 145-161; Davies, N. W., Lux, H. D., and Morad, M. (1988). Site and mechanism of activation of proton-induced sodium current in chick dorsal root ganglion neurones. J Physiol (Lond) 400, 159-187). Although the molecular identity of the channels responsible for these currents is unknown, they have been hypothesized to be acid-sensing members of the DEG/ENaC protein family based on their ion selectivity, voltage insensitivity, and expression pattern (Babinski, K., Le, K. T., and Sxc3xa9guxc3xa9la, P. (1999). Molecular cloning and regional distribution of a human proton receptor subunit with biphasic Adfunctional properties. J. Neurochem 72, 51-57; Bassilana, et al. (1997); de Weille, J. R., Bassilana, F., Lazdunski, M., and Waldmann, R. (1998). Identification, functional expression and chromosomal localisation of a sustained human proton-gated cation channel. FEBS Lett 433,257-260; Lingueglia, et al. (1997); Waldmann, R., Bassilana, F., de Weille, J., Champigny, G., Heurteaux, C., and Lazdunski, M. (1997). Molecular cloning of a non-inactivating proton-gated Na+ channel specific for sensory neurons. J Biol Chem 272, 20975-20978). The acid-sensing ion channels include the brain Na+ channel 1 (BNC1) and its differentially spliced isoform MDEG2 (Garcxc3xada-Anoveros, J., Derfler, B., Neville-Golden, J., Hyman, B. T., and Corey, D. P. (1997). BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels. Proc Natl Acad Sci USA 94, 1459-1464; Lingueglia, et al. (1997); Price, M. P., Snyder, P. M., and Welsh, M. J. (1996). Cloning and expression of a novel human brain Na+ channel. J Biol Chem 271, 7879-7882; Waldmann, R., Champigny, G., Voilley, N., Lauritzen, I., and Lazdunski, M. (1996). The mammalian degenerin MDEG, an amiloride-sensitive cation channel activated by mutations causing neurodegeneration in Caenorhabditis elegans. J Biol Chem 271, 10433-10436), the acid-sensing ion channel (ASICxcex1) and its differentially spliced isoform ASICxcex2 (Chen, C.-C., England, S., Akopian, A. N., and Wood, J. N. (1998). A sensory neuron-specific, proton-gated ion channel. Proc Natl Acad Sci USA 95, 10240-10245; Waldmann, et al. (1997)), and the dorsal root acid-sensing ion channel (DRASIC) (Mammalian neuronal DEG/ENaC channels have several names. The names of the three channels, listed in the order of their publication are: (1) BNC1, MDEG, BNaC1, ASIC2, and the splice variant MDEG2; (2) BNaC2, ASICxcex1, ASIC1, and the splice variant ASICxcex2; and (3) DRASIC and ASIC3.)(Babinski, et al. (1999); de Weille, et al. (1998); Waldmann, et al. (1997)). BNC1, MDEG2, ASICxcex1, and DRASIC are expressed in the central nervous system (Chen, et al. (1998); Lingueglia, et al. (1997); Olson, T. H., Riedl, M. S., Vulchanova, L., Ortiz-Gonzalez, X. R., and Elde, R. (1998). An acid sensing ion channel (ASIC) localizes to small primary afferent neurons in rats. Neuron 9, 1109-1113; Waldmann, et al. (1997)). ASICxcex1, ASICxcex2, DRASIC and MDEG2 are expressed in sensory neurons of the dorsal root ganglia (DRG) (Babinski, et al. (1999); Chen, et al. (1998); Olson, et al. (1998); Waldmann, et al. (1997)).
For the foregoing reasons, there is a need for determination and characterization of the roles of FMRFamide and FMRFamide-related peptides in potentiation of DEG/ENaC channels, especially the acid-sensing ion channels.
The present invention identifies a family of proteins that potentiates the effects of a group of acid-sensing ion channels (DEG/ENaC) which are responsible for pain associated with pain from ischemia and inflammation and certain other physiological effects.
An object of the invention is an assay for screening compositions which effect the acid-sensing ion channels.
Another object of the invention is an assay for screening analgesics.
A further object of the invention is a kit which can be used for performing the assay.
Yet another object of the invention is drug compositions identified by the screening assay.
These and other objects, features, and advantages will become apparent after review of the following description and claims of the invention.
FMRFamide and FMRFamide-like peptides modulate acid-activated currents. The present invention provides an assay for screening compositions to identify those which are agonists, antagonists, or modulators of acid-sensing channels of the DEG/ENaC family. This assay can be especially useful for determining analgesics. The assay comprises administering the composition to be screened to cells expressing acid-gated channels and then determining whether the composition inhibits, enhances, or has no effect on the channels when acid is introduced. The determination can be performed by analyzing whether a current is sustained by the cells in the presence of the composition and the acid. This current can be compared to that sustained by the FMRFamide and related peptides. This assay can also be provided in kit form.